Poster abstracts

Poster number 116 submitted by Kyle Piccolo

Structural analysis of the sulforhodamine B binding aptamer (SRB-2)

Kyle Piccolo (Dept. of Biochemistry, University of Waterloo), Thorsten Dieckmann (Dept. of Biochemistry, University of Waterloo)

Abstract:
Aptamers are nucleic acid sequences selected for in vitro that non-covalently bind a ligand. An aptamer for a desired ligand is selected for by systematic evolution of ligands by exponential enrichment (SELEX), yielding a nucleic acid sequence with high affinity and selectivity for its target (1). As a result of these properties, aptamers have several potential applications in therapeutics, drug delivery, biosensing, analytical chemistry and live cell imaging (2,3). The sulforhodamine B binding aptamer (SRB-2) is an RNA aptamer that was created via SELEX and subsequently optimized to a 54nt sequence. The authors also determined possible sites of binding interactions for SRB-2 by phosphorothioate mapping (4). SRB-2 has also been successfully implemented in live cell imaging experiments including co-localization with the dinitroaniline-binding aptamer (DNB) (5). As SRB-2 is a potentially useful model system, we wish to determine a solution structure for the aptamer by NMR spectroscopy. 1H NMR and 2D NOESY experiments were performed on SRB-2 and when titrated with sulforhodamine B, a gradual change in the chemical shift and peak intensity of several hydrogen atoms and NOEs were observed until saturation. This indicates that the ligand is binding and it is therefore reasonable that a solution structure complete with restraints, may be determined through further NMR analysis.

References:
(1) Kenan, D.J. and Keene, J.D. 1999. In vitro selection of aptamers from RNA libraries. Methods Mol. Biol. 118: 217–231.
(2) Keefe, A.D.; Pai, S. and Ellington, A. 2010. Aptamers as therapeutics. Nature Reviews Drug Discovery 9, July 2010. pp. 537-550
(3) Tombelli, S.; Minunni, N. and Mascini, M. 2005. Analytical applications of aptamers. Biosensors and Bioelectronics 20. pp. 2424–2434
(4) Holeman, L. A.; Robinson, S.L.; Szostak, J.W. and Wilson, C. 1998. Isolation and characterization of fluorophore binding RNA aptamers. Folding and Design, 3(6): 423-431.
(5) Arora, A.; Sunbul, M. and Jäschke, A. 2015. Dual-colour imaging of RNAs using quencher and fluorophore-binding aptamers. Nucl. Acids Res. 2015 Volume.

Keywords: Aptamer, SRB-2, NMR NOESY